Mutant proteins and methods for their production

ABSTRACT

The present invention relates to mutant transmembrane proteins which have increased conformational stability when compared to their parent protein, methods of selection and production. In particular the invention relates to mutant transmembrane proteins which are mutated in or in the proximity of the transmembrane alpha helices or in a kinked region or in an alpha-helix adjacent to a kink. The mutant transmembrane proteins have use in crystallisation studies and also in screening to identify compounds for use in drug discovery and therapy.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/893,867, filed Feb. 12, 2018, which is a continuation of U.S. patentapplication Ser. No. 14/776,908, filed Sep. 15, 2015, which is anational stage filing under 35 U.S.C. § 371 of international applicationPCT/GB2014/050757, filed Mar. 13, 2014, which was published under PCTArticle 21(2) in English, and claims the benefit under 35 U.S.C. §119(e) of U.S. provisional patent application Ser. No. 61/790,592, filedMar. 15, 2013, the disclosure of each of which is incorporated byreference herein in its entirety.

FIELD

The present invention relates to mutant transmembrane proteins whichhave increased conformational stability when compared to their parentprotein, methods of selection and production. In particular theinvention relates to mutant transmembrane proteins which are mutated inor in the proximity of the transmembrane alpha helices or in a kinkedregion or in an alpha-helix adjacent to a kink. The mutant transmembraneproteins have use in crystallisation studies and also in screening toidentify compounds for use in drug discovery and therapy.

BACKGROUND TO THE INVENTION

Structure determination of eukaryotic integral membrane proteins ischallenging and techniques and strategies developed recently hasunderpinned GPCR crystallization, including the development ofreceptor-T4 lysozyme (T4L) fusions (1,2), conformationalthermostabilisation of GPCRs (3-7), and the use of antibody fragments(8-10).

However, the key component for successful crystallization is thestability of the GPCR during purification and crystallization (11).Techniques such as the addition of high affinity ligands to receptor-T4Lfusions, and systemic mutagenesis coupled to a thermostability assay (3,4, 6, 7) have improved the stability of GPCRs in detergent solution,detergent-stability being an essential prerequisite to purification andcrystallisation. The latter approach locks the receptor in a particularconformation which allows successful crystallization as described inWO2008/114020 and WO2009/071914, and also has the advantage that thecrystal structure of the GPCR bound to ligands that bind only veryweakly can be determined (12,13). As described in WO2009/071914 it wasfound that stabilising mutations identified in one GPCR could betransferred to another GPCR by aligning the amino acid sequence thusgenerating a second GPCR with increased stability. However the positionsof the stabilising mutations were not located in a common motif orregion but instread scattered throughout the GPCR (the turkeyβ1-adrenergic receptor, human adenosine receptor, rat neurotensinreceptor and human muscarinic receptor).

In recent years most success in obtaining crystal structures of membraneproteins has been for bacterial proteins (14) since these are easier tooverexpress using known techniques in Escherichia coli than eukaryoticmembrane proteins (15, 16) and are more likely to exhibit stability indetergent solution. In contrast eukaryotic membrane proteins often havepoor stability in detergent solutions which severely restricts the rangeof crystallisation conditions that can be explored. Although thestructures of over 300 unique polytopic integral membrane proteins havebeen determined (blanco.biomol.uci.edu/), less than 10% are eukaryoticand approximately half were purified from natural sources and are stablein detergent solutions.

Transmembrane transporters are similar to GPCRs because they exist in atleast two distinct conformations, with the substrate binding siteaccessible to either the extracellular environment (outward--open) or tothe cytoplasm (inward-open), with a number of potential intermediateoccluded states where the substrate cannot dissociate to either side ofthe membrane (40). Indeed, the structures of many bacterial transportershave been determined that fit into the above scheme and, at least in thecase of Mhp1 (41) and LeuT (26) different conformations of the sametransporter have also been described. Transmembrane transporters havebeen less widely studied than GPCRs but nevertheless are highly relevantin human physiology and disease. They represent valuable targets in drugdiscovery and development of therapeutics, for example the monoaminetransporters are key targets for therapeutic intervention in a widerange of CNS disorders and as primary targets for drugs of abuse such ascocaine and amphetamines (17, 18). Two of the most widely prescribeddrugs fluoxetine (Prozac) and omeprazole (Prilosec) target membranetransporters, and there is a need in the art to understand further thestructure and function of transmembrane transporters and their role indisease to meet the demand for new therapies targeting CNS disorders.

Current methodologies for the crystallisation of transporters haverelied on the identification of those transporters that are sufficentlystable for purification and crystallisation (19) which has allowed thestructure determination of many transporters from different families,but the majority of the structures are of bacterial proteins (20). Tofully understand inhibitor binding and the mechanism of transport of themammalian transporters it is essential to determine their structures.However the mammalian transporters are difficult targets for stucturalstudies due to low levels of functional expression and only a proportionof the expressed protein is correctly folded (21, 22). Heterologousexpression of the cocaine-sensitive rat serotonin transporter (SERT),GABA (GAT) and norepinephrine (NET) is possible in baculovirus systemshowever functional expression levels are low and only a proportion ofthe expressed protein is correctly folded according to the binding ofradiolabelled inhibitors (23, 24).

The SLC6 transporter is a sub-class of the neurotransmitter sodiumsymporter family (NSS) (25) and plays an important role in regulatingneurotransmitter concentrations in the peripheral and central nervoussystem by re-uptake into the presynaptic nerve termini. Mammalian SLC6is characterised by 12 transmembrane helices with a large extracellularloop between transmembrane helices 3 and 4 (TM3 and TM4) that isinvariably N-glycosylated. Structural studies on this family oftransporters has focused on bacterial homologues that are extremelystable, such as LeuT (26).

Lactose permease of E. coli (LacY) has been the focus of a number ofstudies relating to structure and functionality and the crystalstructure has been solved using a mutant (C154G) which renders theprotein unable to undergo the structural changes required for transportof sugar across the membrane. The mutation (Cys154 to Gly) causes a morecompact structure and decreased conformational flexibility with improvedthermostability and little tendency to aggregate. It was also observedthat the conformational change caused little or no effect on ligandbinding (38). These studies demonstrate that it is possible to obtain aconformationally thermostabilised transporter membrane protein by way ofmutation which is suitable for crystallisation. (39). Although thesestudies are of importance for Lactose Permease they do not comment onthe wider problem of how to reliably and efficiently solve the crystalstructures of other transporter proteins and GPCRs. Therefore there is aneed for a common strategy to produce confomationally stable proteinsthat have use in crystallisation studies and stucture determination.Since the provision of conformationally stable mutants of transportersand/or GPCRs and subsequent screening is time consumming, there is aneed for methods that are more efficient and reduce the time taken toproduce a conformationally stable mutant.

In view of the difficulty in obtaining high quality crystal structuresof mammalian proteins due to poor stability and expression and the lownumbers of solved crystal structures, there is a need to produce newmethods and techniques that overcome the above-mentioned problems,particularly for membrane transporters which as yet have not benefitedfrom the intensity of research seen for GPCRs.

The cocaine-sensitive rat serotonin transporter (SERT) is a member ofthe SLC6 sub-class of the neurotransmitter sodium symporter family (NSS)and transports the neurotransmitter serotonin from synaptic spaces intopresynaptic neurons thereby terminating the action of serotonin. SERThas been well characterised in terms of physiological function and isthe target of many antidepressant medications, however its crystalstructure remains to be determined. SERT is known to be unstable indetergent solution (27) making it a challenging target forthermostabilisation studies, however the availability of a high affinityradiolabelled ligand [¹²⁵I] RTI55 (β-CIT) allows dicrimination betweenfunctional and misfolded protein making the protein a suitable candidatefor thermostabilisation studies.

The inventors have applied the conformational thermostabilisationapproach recently used for GPCRs to the cocaine-sensitive rat serotonintransporter (SERT) to improve conformational stability and tolerance indetergent. They found that when particular regions in or nearby thealpha helices of the transmembrane regions of integral membrane proteinsare mutated, this results in a higher proportion of conformationallystable mutants. This discovery has significance for structuredetermination of other related membrane proteins since it is possiblethat mutations in specific regions of a membrane protein can be appliedacross a range of membrane proteins having similar three-dimensionalstructures, thereby improving the probability of obtainingconformationally stable mutants for use in crystallisation.

SUMMARY OF THE INVENTION

According to a first aspect of the invention there is provided a mutanttransmembrane protein which has increased conformational stabilitycompared to its parent transmembrane protein, wherein the one or moremutations are located at the interfaces between transmembranealpha-helices, or in a kinked region or in an alpha-helix adjacent to akink.

According to a further aspect of the invention there is provided amethod of selecting a mutated transmembrane protein comprising the stepsof;

-   -   a) Providing one or more mutants of a parent transmembrane        protein wherein the mutations are at the interfaces between        transmembrane alpha-helices, or in a kinked region or in an        alpha-helix adjacent to a kink.    -   b) Contacting the mutated transmembrane protein with a ligand    -   c) Determining the thermostability of the mutated transmembrane        protein    -   d) Identifying those mutants that exhibit increased        conformational thermostability compared to the parent        transmembrane protein

According to a further aspect of the invention there is provided amethod of producing a mutated transmembrane protein comprising carryingout the steps a) to d) and,

-   -   e) Identifying the position of one or more of the mutated amino        acid residues in those mutants that exhibit increased        conformational stability, and    -   f) Synthesising a mutant transmembrane protein which comprises        the mutated residues identified in step e).

According to a further aspect of the invention there is provided amethod of selecting a binding partner of a mutated transmembraneprotein, the method comprising the steps of

-   -   a) providing a mutant transmembrane protein which has increased        conformational stability and/or is functionally inactive        compared to its parent transmembrane protein, wherein the one or        more mutations are located at the interfaces between        transmembrane alpha-helices, or in a kinked region or in an        alpha-helix adjacent to a kink.    -   b) contacting the mutant transmembrane protein with one or more        compounds    -   c) determining whether the one or more compounds bind to the        mutant transmembrane protein    -   d) isolating one or more compounds.

According to a further aspect of the invention there is provided amutant transmembrane protein obtained by the methods of the previousaspects.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B Optimisation of SERT expression by transient transfection inT-Rex-HEK293 Cells.

(FIG. 1A) The amount of tetracycline used to induce cells transientlytransfected with SERT-mCherry was tested to give good cell surfaceexpression with minimal intracellular expression, as defined by confocalmicroscopy. Examples of cells depicted were either induced with 1.2μg/ml tetracycline (left hand panel) or 0.8 μg/ml tetracycline(right-hand panel). Cells were transfected with 0.1 mg of plasmid DNAper 50,000 cells and were induced for 48 hours. (FIG. 1B) TheDNA:transfection reagent ratio for maximal correctly localisedexpression of SERT. 50,000 cells were transfected with a range of DNAconcentrations. Cells were induced with 0.8 μg/ml tetracycline for 48hours, examined using a confocal microscope and the percentage of plasmamembrane expression of SERT was estimated by eye.

FIGS. 2A-2B Development of a thermostability assay for SERT.

(FIG. 2A) The thermostability of [¹²⁵I]-RTI55-bound SERT was determinedafter solubilisation in three different concentrations of DDM (finalconcentrations in %): squares, 0.01%; triangles, 0.1%; circles, 1%. Thedifference in thermostability is likely a consequence of the degree ofdelipidation of the transporter that increases as the amount ofdetergent increases. Each data point (±SEM) was obtained in duplicatefrom an equivalent of 50,000 cells from a tetracycline-induced stablecell line T-Rex-SERT. (FIG. 2B) Thermostability assays of SERT-His10expressed in the stable cell line T-Rex-SERT (circles) and SERT-mCherrytransiently transfected into T-Rex-HEK293 cells (squares). Cells weresolubilised with 0.1% DDM after the addition of 1 nM [¹²⁵I]-RTI55; theapparent Tm of both samples of SERT was 28° C. All the reactionscontained the equivalent of 50,000 cells per data point. The results arefrom a single experiment performed in duplicate (±SEM).

FIGS. 3A-3C Thermostabilisation of SERT

(FIG. 3A) Comparison of [¹²⁵I]-RTI55 bound to 554 detergent-solubilisedAla/Leu mutants at either 4° C. or after heating at 30° C. for 30minutes. The data relating to thermostability (the heated samples) havebeen normalized to the amount of wild-type SERT remaining after heating(40%; horizontal dashed line). The expression level of wild-type SERT isindicated by the vertical dashed line and the grey column (0-200 dpm)represents non-specific binding of [¹²⁵I]-RTI55 to the parentalT-REx-HEK293 cell line (no SERT). Each data point represents binding tothe equivalent of 50,000 cells measured in duplicate (estimated error is±20% in dpm). (FIG. 3B) The optimally thermostabilised SAH mutants wereengineered by combining the best thermostabilising mutations (asindicated) which resulted in an increased apparent T_(m) aftersolubilisation in 0.1% DDM. (FIG. 3C) Amino acid sequence of SERT (SEQID NO: 1) showing the positions of the thermostabilising mutations(hatched) that consistently gave >1° C. increase in T_(m) compared towild-type SERT; residues in bold and hatched were used to stabilizeeither SAH6 and/or SAH7. N-linked glycosylation sites are indicated bythe hexagons, a putative disulphide bond is shown as a grey line and theN-terminal and C-terminal fusion partners are shown (c-Myc tag and thefluorescent reporter protein mCherry) are indicated.

FIG. 4 Alternative amino acid residues for SERT thermostabilisation.

Amino acid residues already identified as being thermostabilising in thepreliminary Ala/Leu scan were changed either to Ala, Gly, Val Leu or Ile(hatched). Where no functional SERT was detected, the amino acid towhich the residue was mutated is given in the single letter code. Barsmarked with an asterisk showed improved thermostability over theoriginal mutation, but expression levels were less than 10% of the wildtype SERT and therefore these mutations were not used further.

FIGS. 5A-5B Detergent stability of the thermostabilised mutants SAH6 andSAH7.

(FIG. 5A) Thermostability curves for [¹²⁵I]-RTI55-bound DDM-solubilisedSAH6 (circles) and SAH7 (squares) compared to wild-type SERT(triangles). The apparent T_(m)s determined from the curves are: SAH6,46° C.; SAH7, 44° C.; wild-type SERT, 28° C. All the data were collectedin a single experiment with measurements performed in duplicate. (FIG.5B) The stability of [¹²⁵I]-RTI55-bound SAH6, SAH7 and wild-type SERTwas compared in 8 different detergents [¹²⁵I]-RTI55 was added tomembranes (final concentration 1 nM) which were then solubilised for 30min on ice in the following detergents (aliphatic chain length inparentheses, final detergent concentration in %): 0.1% DDM (C12), 1% DDM(C12), 0.4% DM (C10), 0.35% FC12 (C12), 0.3% LDAO (C12), 0.6% Hega-10(C10), 0.5% NG (C9), 0.6% NM (C9), 0.83% OG (C8). Thedetergent-solubilised samples where then heated at 30° C. for 30 minbefore determining the amount of SERT remaining in relation to control(incubated on ice). Results are from a single experiment performed induplicate (±SEM) with the equivalent of 50,000 cells per data point.

FIG. 6 Inhibitor and substrate affinities for SAH6 and SAH7 mutantscompared with wild-type SERT.

Apparent K_(i) values were determined from competition binding curves(FIGS. 7A-7C) and are plotted as the change in affinities with respectto wild-type SERT (ΔpK_(i)). Competition assays were performed onmembranes using a final concentration of 0.2 nM [¹²⁵I]-RTI55 andapparent K values determined using the following apparent KD values for[¹²⁵I]-RTI55 binding (FIGS. 7A-7C): SAH6, 3.7±0.7 nM; SAH7, 1.2±0.5 nM;wild-type SERT, 3.7±2.2 nM. Error bars are proportional to the SEM fromthe original measurements.

FIGS. 7A-7C Competition curves for SAH6 and SAH7.

(FIG. 7A) Competition assays were performed on membranes using a finalconcentration of 0.2 nM [¹²⁵I]-RTI55 and apparent K_(i) values (FIG. 7B)were determined using the following apparent KD values for [¹²⁵I]-RTI55binding (FIG. 7C): SAH6, 3.7±0.7 nM; SAH7, 1.5±0.6 nM; wild-type SERT,3.7±2.2 nM. All results were obtained from two independent experimentsperformed in duplicate (±SEM) . For all graphs: wild type SERT,triangles; SAH6, circles; SAH7, squares.

FIGS. 8A-8B Thermostabilised mutants do not transport SHT.

(FIG. 8A) [³H]5HT uptake assays were performed on tetracycline-inducedstable T-REx cell lines expressing SAH6-GFP, SAH7-GFP, and SERT-GFP,with non-specific uptake determined upon addition of 10 mM cocaine andthe results baseline corrected. The results are from a single experimentperformed in triplicate (±SEM) with approximately 100,000 cells per datapoint. (FIG. 8B) Confocal microscope images of cells expressingtransporter-GFP fusions used in the uptake assays in (FIG. 8A). Nofluorescence was detected in parental T-REx-HEK293 cells. SAH6-GFP,SAH7-GFP and SERT-GFP were all capable of binding [¹²⁵I]-RTI55-bindingwith high affinity (FIGS. 7A-7C).

FIG. 9 Amino acid sequence alignment between Rattus norveqicus SERT andAquifex aeolicus LeuT (19% identity).

Identical residues (*) and similar residues (.) are indicated below thealigned amino acid sequences (SERT, SEQ ID NO: 1; LeuT, SEQ ID NO: 2).The transmembrane alpha-helices in LeuT (as defined by the structure)are shown as grey shading, with unwound regions as a grey line, andother alpha-helices are shown as hatched bars. The positions of the 11thermostable mutations are shown either as hatched (present in SAH6and/or SAH7) or hatched and shaded (other thermostabilising mutations).

FIGS. 10A-10C Equivalent positions in LeuT of the thermostabilisingmutations in SAH6.

A cartoon of LeuT (PDB code 3GWU) is shown in black and white (Nterminus light shade, C-terminus dark shade) with bound leucine (hatchedspheres), Na+ ions (spheres) and sertraline (sticks). Shaded spheresindicate amino acid side chains in LeuT that are equivalent to thethermostabilising mutations found in SAH6 (in parentheses): Leu25(L99A); Ala195 (G278A); Gly416 (A505L). LeuT is shown either viewed fromthe extracellular surface of the membrane (FIG. 10A) or parallel to themembrane plane (FIG. 10B). Portions of the structures have beenhighlighted (boxes, FIG. 10C) to depict the position of the residues inrelation to helix-helix interfaces.

FIGS. 11A-11C Equivalent positions in LeuT of the thermostabilisingmutations in SAH7.

A cartoon of LeuT (PDB code 3GWU) is shown in black and white (Nterminus light shade, C-terminus dark shade) with bound leucine (hatchedspheres), Na+ ions (spheres) and sertraline (sticks). Shaded spheresindicate amino acid side chains in LeuT that are equivalent to thethermostabilising mutations found in SAH7 (in parentheses): Leu322(L405A);Ile410 (P499A); Gly416 (A505L). LeuT is shown either viewed fromthe extracellular surface of the membrane (FIG. 11A) or parallel to themembrane plane (FIG. 11B). Portions of the structures have beenhighlighted (boxes, FIG. 11C) to depict the position of the residues inrelation to helix-helix interfaces.

FIGS. 12A-12D Amino acid residues in LeuT equivalent to thethermostabilising mutations in SAH6. (FIG. 12A) The structure of LeuT(PDB code 3GWU) is depicted in black and white (N terminus, light shade;C-terminus, dark shade) with the side chains in equivalent positions tothe thermostabilisation mutations in SAH6 shown as shaded spheres. Theview is from the extracellular surface perpendicular to the membraneplane. (FIGS. 12B-12D) The mutations are found at helix-helix interfacesand often at the sites of kinks or unwound regions; (FIG. 12B)Gly416LeuT (A505L^(SAH6)); (FIG. 12C) Leu25LeuT (L99A^(SAH6)); (FIG.12D) Ala195^(LeuT) (G278A^(SAH6)). Additional views of the mutations arein FIGS. 10A-10C and for details of SAH7 mutations, see FIGS. 11A-11C.

FIGS. 13A-13D Crystallisation of thermostabilised SERT

(FIG. 13A) Crystallisation drop viewed under white light. (FIG. 13B) Thesame area of the crystallisation drop viewed under UV light whereprotein crystals appear white on a dark background. (FIG. 13C) Onecrystal mounted on a loop for collection of X-ray diffraction patterns.(FIG. 13D) One X-ray diffraction pattern showing diffraction spots to 8Å resolution (the ring shows the 7.5 Å diffraction limit).

DETAILED DESCRIPTION OF THE INVENTION

According to a first aspect of the invention there is provided a mutantmembrane protein which has increased conformational stability comparedto its parent membrane protein, wherein the one or more mutations arelocated at the interfaces between transmembrane alpha-helices, or in akinked region or in an alpha-helix adjacent to a kink.

References herein to “membrane protein” refer to a protein that isattached to or associated with a membrane of a cell or organelle.Examples of membrane proteins include GPCRs, T-cell receptors, growthfactor receptors, transmembrane ion channels, transmembranetransporters, a ligand-gated transmembrane ion channel, a voltage-gatedtransmembrane ion channel, an enzyme, a carrier protein or an ion pump.Membrane proteins may comprise more than one polypeptide chain.

For example the membrane protein may be an integral membrane proteinthat is permanently integrated into the membrane and can only be removedby using detergents, non-polar solvents or denaturing agents thatphysically disrupt the lipid bilayer.

References to “transmembrane protein” herein refer to an integralmembrane protein which extends from one side of the membrane through tothe other side of the membrane, thus spanning the entire membrane.

References to “transmembrane transporters” herein refer to transmembraneproteins which are responsible for the transport of ions, smallmolecules or macromolecules across a membrane. Suitable transmembranetransporters include monamine transporters, for example thecocaine-sensitive rat serotonin transporter (SERT), the dopaminetransporter (DAT) and the norepinephrine transporter (NET).

In one embodiment the membrane protein is an integral membrane proteinor a transmembrane protein.

In another embodiment the membrane protein is a transmembranetransporter or a GPCR. In another embodiment the transmembranetransporter is a monamine transporter. In a further embodiment thetransmembrane transporter is a member of the SLC6 sub-class of theneurotransmitter sodium symporter family (NSS).

In a further embodiment the transmembrane transporter is thecocaine-sensitive rat serotonin transporter (SERT).

The membrane protein may be derived from any source, for example aeukaryotic source, a prokaryotic source, or from cell-free systems.

In one embodiment the membrane protein is mammalian and derived fromrat, mouse, rabbit, dog, or non-human primate, man, chicken or turkey.

Suitable GPCRs are well known in the art and include those listed inFoord et al (2005) Pharmacol Rev. 57, 279-288, incorporated herein byreference, (which is periodically updated atiuphar-db.org/DATABASE/ReceptorFamiliesForward?type=GPCR).

Reference to “mutant membrane protein” herein refers to a membraneprotein with a different genotype to the wild type (or parent) membraneprotein. Such a mutant may also result in a different phenotypicdifference.

Reference to “Parent membrane protein” herein refers to a protein whichretains a functional activity of the naturally occurring protein.Functional activity may be for example, ligand binding and/or thetransport of ions, small molecules or macromolecules across themembrane. The parent membrane protein may be more or lessconformationally stable than the mutant membrane protein.

Mutants of the membrane protein may be produced by any suitable methodwhere each amino acid of the parent membrane protein is independentlychanged to a different amino acid residue. Molecular biologicaltechniques for cloning and engineering genes and cDNAs, for mutatingDNA, and for expressing polypeptides from polynucleotides in host cellsare well known in the art as exemplified “Molecular cloning, alaboratory manual”, third edition, Sambrook, J. & Russell, D. W. (eds),Cold Spring Harbor Laboratory press, Cold Spring Harbor, N.Y.,incorporated herein by reference.

Mutations may be made in any part of the membrane protein, for examplein the part which spans the membrane.

In one embodiment the mutant membrane protein is produced by alaninescanning mutagenesis, a technique well known in the art. Here eachselected amino acid is replaced in turn with alanine to produce a seriesof mutants suitable for screening. If the selected amino acid is Ala itis replaced with Leu or Gly. Selected regions of the membrane proteinmay be chosen for alanine mutagenesis, for example amino acid residues49 to 603 inclusive for SERT.

In another embodiment the mutant membrane protein is produced by randommutagenesis, which may be in the whole of the protein or in a selectionpart. Such techniques are well known in the art (Asubel et al, CurrentProtocols in Molecular Biology, John Wiley & Sons, New York 2000).

In one embodiment the mutant membrane protein comprises one or moremutations located at the interfaces between transmembrane alpha-helices,or in a kinked region or in an alpha-helix adjacent to a kink. Themutant membrane protein may also comprise one or mutations which are atthe sites of unwound regions of the alpha-helix.

References to “transmembrane alpha-helices” herein refer to the helicalthree-dimensional amino acid structure found within the transmembranespanning domains of a membrane protein. Transmembrane alpha-helices maybe in the form of a single alpha-helix spanning the membrane, a pair ofhelices or any number of helices passing through the membrane typicallyranging from one to twenty-four with common arrangements typified byseven helices passing through the membrane, as in GPCRs, or twelvehelices passing through the membrane as in many transporters.

Reference to “interfaces” between transmembrane alpha-helices refers tothe regions which are between the strands of the alpha-helix as arrangedin the membrane, also known as helix-helix interfaces. Specificallyinterfaces are defined as regions were the atoms of amino acids residuesin two or more separate helices are within van der Waals radii of eachother.

Reference to “kinked region” herein refers to the regions whichrepresent a turn of the alpha-helix as arranged in the membrane suchthat as a result of the kink the helix no longer has a linearconformation but instead comprises a bend or curve.

In one embodiment the mutant membrane protein comprises one or morereplaced amino acids compared to the parent membrane protein. In anotherembodiment the mutant membrane protein comprises two, three, four, five,six or seven replaced amino acids compared to the parent membraneprotein. In a further embodiment the mutant membrane protein comprisesbetween two and four replaced amino acids compared to the parentmembrane protein. In a further embodiment the mutant membrane proteincomprises three or four replaced amino acids compared to the parentmembrane protein.

In another embodiment the mutant membrane protein is thecocaine-sensitive rat serotonin transporter (SERT) and comprises atleast one of the mutations selected from P499A, A505L, G113A, L99A,G278A, A169L, F311A, G115A, L405A and L406A.

In another embodiment the mutant cocaine-sensitive rat serotonintransporter (SERT) and comprises two, three or four of the mutationsselected from P499A, A505L, G113A, L99A, G278A, A169L, F311A, G115A,L405A and L406A.

In another embodiment the mutant membrane protein is thecocaine-sensitive rat serotonin transporter (SERT) and comprises atleast one of the mutations selected from G278A, A505L, L99A and P499A.

In a further embodiment the mutant membrane protein is thecocaine-sensitive rat serotonin transporter (SERT) and comprises themutations L99A, G278A and A505L.

In a further embodiment the mutant membrane protein is thecocaine-sensitive rat serotonin transporter (SERT) and comprises themutations L405A, P499A and A505L.

In another embodiment the mutant membrane protein comprises at least oneor more mutations which are at the corresponding amino acid positions ofP499A, A505L, G113A, L99A, G278A, A169L, F311A, G115A, L405A and L406Aas defined in the amino acid sequence of SERT shown in FIG. 9.

By “corresponding amino acid position or positions” we mean the positionin the amino acid sequence of SERT which aligns to the position in theamino acid sequence of the membrane protein when the sequence of SERTand a membrane protein are compared by alignment using, for example,MacVector and the Clustal W program. Such alignment techniques are knownin the art and appreciated by the skilled person.

In a further embodiment the mutant membrane protein comprises at leastone or more mutations which are within a window of amino acids eitherside of the of the position which corresponds to the amino acidpositions of one or more of P499A, A505L, G113A, L99A, G278A, A169L,F311A, G115A, L405A and L406A.

A “window of amino acids” refers to a window of i plus or minus fouramino acid residues where i is defined as the stabilising mutation.

The stability of the mutant membrane protein is compared to the parentor wild type membrane protein to establish if the presence of the one ormore mutations results in an increase in conformational stability.

Reference to “conformational stability” herein refers to theconformation adopted by a membrane protein that results in an improvedstability with respect to any one of biological activity of the membraneprotein such as binding activity, a signalling pathway modulationactivity, a transmembrane transporting activity or an enzyme activity.

Increased conformational stability may be observed when the mutantmembrane protein is bound to a ligand. A ligand may function as aninhibitor, an agonist or an antagonist. Ligand binding may cause themutant membrane protein to reside in a particular conformation, forexample with respect to GPCRs, an agonist or antagonist conformationdepending on whether the ligand functions as an agonist or antagonist.Thus the presence of the ligand may be considered to encourage the GPCRto adopt a particular conformation. As a function of the ability ofmembrane transporters to facilitate the vectorial movement of substratesacross biological membranes, they can exist in two differentconformations known as the outward-open conformation and the inward-openconformation. Ligand binding may influence which conformation themembrane transporter resides in. In this respect, ligand binding thatblocks or prevents transport of substances across the membrane functionsas an inhibitor. Antibodies, including fragments and derivativesthereof, may function as ligands and induce conformational change inGPCRs and membrane transporters. Antibodies may be specific for aparticular conformation of a GPCR or membrane transporter.

In one embodiment the mutant membrane protein is a membrane transporterand is conformationally stabilised in a single conformation such as theoutward-open conformation or the inward-open conformation when bound toa ligand.

In another embodiment the mutant membrane transporter is SERT and isconformationally stabilised in the outward-open conformation when boundto a ligand, for example RT155 (β-CIT).

The ligand may be detectably labelled in accordance with techniquesknown in the art. For example the ligand is radiolabelled or conjugatedto a fluorescent label.

The mutant membrane protein may have an increased stability to any oneor more of heat, a detergent, a chaotropic agent and extreme of pH.

Increased stability to heat (i.e. thermostability) can be readilydetermined by measuring ligand binding or by using spectroscopictechniques such as fluorescence, CD or light scattering at a particulartemperature. In one embodiment the thermostability of a mutant membraneprotein is determined by measuring the T_(m) (the temperature at which50% of the mutant membrane protein is inactivated under certainconditions for a given period of time (e.g. 30 minutes). Mutant membraneproteins having a higher thermostability have higher T_(m) values whencompared to the parent membrane protein.

To determine increased stability to a detergent or a chaotrope, themutant membrane protein is incubated for a defined time in the presenceof a test detergent or a test chaotropic agent and the stability ismeasured using for example ligand binding or a spectroscopic method asdiscussed above.

Suitable detergents for solubilisation of the membrane protein and/ormutant membrane protein and for measuring conformational stability areknown to the skilled person in the art and include for example,dodecylmaltoside (DDM), CHAPS, octylglucoside (OG) and many others.

To determine increased stability to an extreme of pH, a typical pH testwould be chosen, for example, in the range 4.5 to 5.5 (low pH) or in therange 8.5 to 9.5 (high pH).

In one embodiment the mutant transmembrane protein has increasedconformational thermostability when compared to the parent membraneprotein.

In another embodiment the mutant transmembrane protein has increasedconformational thermostability compared to the parent membrane proteinby at least 1° C.

According to a further aspect of the invention, there is provided amethod of selecting a mutated transmembrane protein comprising the stepsof;

-   -   a) Providing one or more mutants of a parent transmembrane        protein wherein the mutations are at the interfaces between        transmembrane alpha-helices, or in a kinked region or in an        alpha-helix adjacent to a kink.    -   b) Contacting the mutated transmembrane protein with a ligand    -   c) Determining the stability of the mutated transmembrane        protein    -   d) Identifying those mutants that exhibit increased        conformational stability compared to the parent transmembrane        protein.

Methods according to steps b) to d) are known in the art and describedin WO2009/071914 incorporated herein by reference. It is appreciatedthat such methods are equally applicable to transmembrane transporters.

In one embodiment increased conformational stability is increasedconformational thermostability.

In another embodiment increased conformational stability is determinedin the presence of detergent.

Methods of providing one or more mutants of a parent transmembraneprotein wherein the mutations are at the interfaces betweentransmembrane alpha-helices, or in a kinked region or in an alpha-helixadjacent to a kink is described above.

Methods of determining thermostability are as described in the methodssection and in example 2.

In one embodiment the mutated parent transmembrane protein is contactedwith a ligand prior to detergent-solubilisation and measurement ofthermostability. Alternatively the mutated parent transmembrane proteinis contacted with a ligand after detergent-solubilisation.

In one embodiment mutants of a parent transmembrane protein withincreased conformational thermostability compared to the parenttransmembrane protein were identified as having an increase in stabilityof at least 1° C.

According to a further aspect of the invention there is provided amethod of producing a mutated transmembrane protein wherein themutations are at the interfaces between transmembrane alpha-helices, orin a kinked region or in an alpha-helix adjacent to a kink, comprisingcarrying out the steps a) to d) and,

-   -   e) Identifying the position of one or more of the mutated amino        acid residues in those mutants that exhibit increased        conformational stability, and

Synthesising a mutant transmembrane protein which comprises the mutatedresidues identified in step e).

Methods for identifying the positions of the mutated amino acidsaccording to step e) are carried out by sequencing techniques known inthe art.

According to a further aspect of the invention there is provided amethod of selecting a binding partner of a mutated transmembraneprotein, the method comprising the steps of

-   -   a) providing a mutant transmembrane protein which has increased        conformational stability and/or is functionally inactive        compared to its parent transmembrane protein, wherein the one or        more mutations are located at the interfaces between        transmembrane alpha-helices, or in a kinked region or in an        alpha-helix adjacent to a kink.    -   b) contacting the mutant transmembrane protein with one or more        compounds    -   c) determining whether the one or more compounds bind to the        mutant transmembrane protein    -   d) isolating one or more compounds.

The mutated transmembrane proteins which have been identified as havingconformational stability have use in the identification of ligands whichbind to the transmembrane protein when it is in a particularconformation. The provision of ligands which bind the transmembraneprotein in a particular conformation are valuable tools for thedevelopment of agents for therapeutic use.

Methods of screening are known in the art and described in WO2008/004223herein incorporated by reference. It is appreciated that such methods ofscreening are equally applicable to transmembrane transporters.

In one embodiment the mutant membrane protein is immobilised onto asolid support.

According to a further aspect of the invention there is provided amutated transmembrane protein obtainable by the methods describedherein.

Methods

All radiolabelled ligands were purchased from Perkin Elmer anddetergents were from Anatrace.

Thermostability Assay

The thermostability of detergent-solubilised [¹²⁵I]-RTI55-bound SERT wasdetermined as previously described for GPCRs (3, 4, 6, 7). Briefly,cells containing unpurified SERT were incubated with 1 nM [¹²⁵I]-RTI55for 30 min on ice, which were then solubilised with detergent (DDM) onice for 30 min before incubation at varying temperatures for 30 min. Theradioligand bound to the membrane protein was separated from freeradioligand by centrifugal gel filtration and the radioligand bound tothe eluted transporter measured by liquid scintillation counting.

Radiolabelled Inhibitor Binding Assay

Saturation binding curves for membrane-bound SERT were obtained using arange of [¹²⁵|]-RTI55 concentrations from 0.13 nM to 160 nM in a 96-wellplate format with non-specific binding being accounted for by incubatingidentical samples with 1 μM cocaine. The samples were incubated for 2hours at 30° C. and then filtered on 96-well glass-fibre plates(Millipore) pre-treated with 200 μl 0.1% polyethyleneimine. The filterswere washed three times with 200 μl ice-cold SERT buffer (100 mM NaCl,20 mM Tris pH 7.4), dried for one hour at 50° C. prior to liquidscintillation counting. Competition binding assays were performed asabove but a range of concentrations of unlabelled ligand was includedand a final concentration of 0.2 nM [¹²⁵I]-RTI55 was used.

[³H]-5HT Uptake Assays

The [³H]-5HT uptake assays were performed with slight modifications tothe method previously described (28). In brief, T-Rex293 cells andT-REx-SERT cells were plated onto poly-L-lysine-coated (1 mg/ml) 24-wellplates, grown to 80% confluency, induced by the addition of 0.8 μg/mltetracycline and grown for 48 hours. The growth medium was aspirated andthe cells washed once with TB buffer (10 mM Hepes pH 7.5, 150 mM NaCl, 2mM KCl, 1 mM CaCl₂, 1 mM MgCl₂). The assays were performed at 25° C.using 1 million cells in 400 μl TB buffer and 2 μM [³H]-5HT andterminated 3 minutes after addition of substrate by three washes ofice-cold TB buffer containing 1 μM paroxetine or 10 μM cocaine. [³H]-5HTwas released by rupturing the cells with 2% SDS, which was quantified byliquid scintillation counting. Non-specific uptake was defined as[³H]-5HT transport in the presence of 10 μM paroxetine or 10 μM cocaine.

EXAMPLES Example 1 cDNA Expression of SERT in HEK293 Cells

The construct C-myc-SERT-mCherry-BioHis10 was developed from the SERTcDNA in plasmid pCGT137 (23) and inserted into the mammalian cellexpression vector pcDNA5/FRT/TO (Invitrogen), which was used for sitedirected mutagenesis and expression. Cells were induced for 48 hourswith 1.2 μg/ml or 0.8 μg/ml tetracycline. 0.1-0.2 μg of plasmid per50,000 T-Rex-HEK293 cells was identified as the optimal amount to ensurethe majority of SERT was expressed at the cell surface as observed byconfocal microscopy (FIG. 1A).

Stable cell lines expressing SAH6-GFP and SAH7-GFP in T-REx-293 cellswere generated by selection with media containing 200 μg/ml zeocin.

Example 2 Development of a Thermostability Assay for SERT

Binding assays using an excess of [¹²⁵I]-RT155 at a concentration of 1nM (5 times the K_(D)) (23) showed that there were on averageapproximately 100,000 copies per transfected cell and that there weresufficient molecules of SERT in 50,000 cells per well of a 96-well plateto perform a single-point thermostability assay in duplicate.Thermostability was determined after solubilisation in three differentconcentrations of DDM (0.01%, 0.1% and 1%) (FIG. 2A).

Thermostability assays usually involved adding the radioligand todetergent solubilised membrane proteins (7), but SERT is only stable indigitonin (29) therefore a different assay was developed where aninhibitor was used to stabilse the transporter. This entailed adding[¹²⁵I]-RTI55 to the T-Rex-HEK293 cells followed by detergentsolubilisation and then the thermostability assay (heating samples atvarious temperatures for 30 minutes). The apparent T_(m) was defined asthe temperature where 50% of the transporter still bound theradiolabelled inhibitor (FIG. 2B). For [¹²⁵I]-RTI55-bound SERT, theapparent T_(m) was 28° C., regardless of how it was expressed in HEK293cells (FIG. 2B). Note that a considerable proportion of thisthermostability is attributable to the bound inhibitor, because theapparent T_(m) of DDM-solubilised SERI without bound [¹²⁵I]-RTI55 couldnot be measured. [¹²³I]-RTI55-bound SERT was also sensitive to theconcentration of DDM present in the assays (FIGS. 2A-2B), with theapparent T_(m) decreasing as the concentration of detergent increases.The most reproducible results with the steepest thermostability curvewere obtained with a final concentration of 0.1% DDM, so this was usedin subsequent assays to determine the thermostability of SERT mutants.The assays were repeated using either [³H]-imipramine or [³H]-paroxetineunder identical conditions, but the binding characteristics of eitherligand in the presence of DDM were unsatisfactory.

Example 3 Generation of SERT Mutants

Systematic alanine-scanning mutagenesis was performed throughout SERIbetween amino residues 49 and 603, with each residue changed to alanineor, if the residue was already alanine, then it was changed to leucine.Mutants were generated by PCR using the QuikChange II methodology(Stratagene) using KOD Hot Start polymerase (Novagen). PCR reactionswere transformed into XL1-competent cells (Stratagene). Mutations werecombined by PCR as above, but using multiple primers. Each SERT-mCherrymutant was sequenced to ensure that only the desired mutation waspresent. A total of 554 mutants were constructed throughout thetransmembrane domains and all loop regions (FIGS. 3A-3C).

The N-terminus and C-terminus were not mutated because these regionswere predicted to be disordered and they are therefore unlikely tocontribute to the thermostability of SERT. Plasmid DNA for eachSERT-mCherry mutant was amplified using a Maxi-prep kit (Qiagen) andtransiently transfected (GeneJuice, Novagen) into adherent mammalianT-REx-293 cells (50% confluent) grown in DMEM media supplemented with10% tetracycline-free FBS and 5 μg/L blasticidin. Expression of mutantswas induced by addition of 0.8 μg tetracycline/ml and incubation at 37°C. for 24 hours. Expression was assessed by fluorescence microscopy toascertain whether the mutant was predominantly either at the plasmamembrane or intracellular. The thermostability of each mutant was thendetermined using a single-temperature thermostability assay and comparedto the thermostability of wild-type SERT. The sample was heated at 28°C. for 30 minutes and approximately 40% of wild-type SERT remainedfunctional. Each batch of mutants tested contained wild-type SERT as acontrol so that the data between different experiments could benormalized (wild-type=40%). Analysis of the results (FIGS. 3A-3C)identified 34 mutations that appeared to improve the thermostability ofSERT, but which did not decrease the levels of expression by more than70%. Interestingly, there was no correlation between the levels ofexpression and thermostability of the mutants, in contrast to in GPCRswhere a weak correlation was sometimes observed (r²=0.2) (11). Of the 34mutations identified, full thermostability curves showed that 10mutations improved the thermostability of SERT by at east 1° C. as shownin Table 1 below.

TABLE 1 Thermostability and expression data for the most thermostableAla/Leu mutants. Expression (%) Cell surface Apparent T_(m) ΔT_(m)Mutation SERT = 100% expression (° C.) (° C.) P499A 65 ** 35 7 A505L 175*** 34 6 G113A 42 ** 33 5 L99A 131 ** 32 4 G278A 39 * 31 3 A169L 31 **30 2 F311A 492 ** 30 2 G115A 327 *** 29 1 L405A 543 *** 29 1 L406A 87 *29 1 The top 34 SERT mutants as estimated from the single pointthermostability assay were re-tested using a 6-point thermostabilitycurve to determine an accurate apparent T_(m) and expressed as animprovement in T_(m) (ΔT_(m)) assuming wild-type SERT had an apparentT_(m) of 28° C. Each mutant was also assessed for cell surfaceexpression as determined by estimation by eye of fluorescence throughoutthe cell upon confocal microscopy: * low expression; ** as wild-typeSERT, *** higher expression than wild-type SERT.

Of these 10 mutations, 7 were in the transmembrane helices and 3 in theextracellular loops (FIGS. 3A-3C). Further mutation of these Ala/Leumutants to other amino acid residues did not significantly improve thethermostability of SERT (FIG. 4).

Combining the thermostabilising mutations in SERT was performed by arational process previously described for the thermostabilisation ofagonist-bound neurotensin receptor and adenosine A2_(A) receptor (3).The best four thermostabilising mutations (P499A, A505L, L99A, G113A)were each combined with each other to make a series of double mutants(Table 2). Of these mutants, the most thermostable were A505L+L99A(SAH4) and A505L+P499A (SANS). These double mutants were then combinedwith the remaining mutants to make triple mutants (Table 2), with themost thermostable being SAH6 (A505L+L99A+G278A) and SAH7(A505L+P499A+L405A) with apparent T_(m)s 16° C. and 18° C. higher thanwild-type SERT

(FIGS. 5A-5B). Further combinations of mutations did not improvesignificantly the thermostability of these mutants (Table 2), so SAH6and SAH7 were identified as the best candidates for structural studiesand were therefore characterized further.

TABLE 2 Thermostability of double and triple mutants. Apparent T_(m)SERT mutation in 0.1% DDM Wild type None 28° C. SERT Double A505L +P499A (SAH5) 39° C. mutants A505L + L99A (SAH4) 43° C. P499A + L99A 35°C. P499A + F311A — A505L + F311A 32° C. L99A + F311A 27° C. TripleA505L + P499A + L405A (SAH7) 44° C. mutants A505L + P499A + A169L 33° C.A505L + P499A + F311A 44° C. A505L + P499A + L99A — A505L + L99A + G278A(SAH6) 46° C. A505L + L99A + F311A 43° C. A505L + L99A + L405A 43° C.A505L + L99A + L406A 44° C. A505L + L99A + N101A 40° C. A505L + L99A +G115A 44° C. A505L + F311A + L99A 40° C. Quadruple A505L + P499A +L405A + F311A 44° C. mutants A505L + P499A + L405A + G115A 45° C.A505L + L99A + G278A + F311A 44° C. A505L + L99A + G278A + G115A 44° C.A505L + L99A + G278A + L405A 44° C. Combinations of SERT mutants testedfor the thermostabilisation of ¹²⁵I-RTI55-bound detergent-solubilisedSERT (apparent T_(m) ± 1° C.).

Surprisingly, when the mutations were mapped to the LeuT structure, allthe mutations in SAH6 and SAH7 are found at the interfaces betweentransmembrane a-helices and, more specifically, in either a kinkedregion or in an a-helix adjacent to a kink. The conserved nature ofamino acid residues that have been mutated to improve thermostability ofSERT suggests that similar mutations in related transporters such as fornorepinephrine (NET) and dopamine (DAT) would also improve theirthermostability, as has been observed when thermostabilising mutationshave been transferred between closely related GPCRs (37).

Example 4 Characterisation of Optimally Stabilised Mutants SAH6 and SAH7

Radioligand binding assays were performed on the thermostabilised SERTmutants as described above. The affinity of SAH6 and SAH7 for[¹²⁵I]-RTI55 in saturation ligand binding assays was found to be largelyunchanged with apparent K_(D)s of 3.8±0.1 nM and 1.3±0.1 nM,respectively, compared to 1.6 nM±0.1 nM for wild-type SERT (FIGS.7A-7C).

Competition binding assays (FIGS. 7A-7C) showed that SAH6 had anapparent affinity for cocaine 17-fold higher than wild-type SERT (FIG.6), whilst there was a small decrease in affinity for ibogaine,imipramine, paroxetine and serotonin (4.5-, 6.7-, 90-, 2.9-fold,respectively). SAH7 showed a similar profile of binding, although theabsolute values differed slightly (FIGS. 7A-7C, FIG. 6).

Both SAH6 and SAH7 were found to be more stable in short chaindetergents that are suitable for crystallography (11) and hence possessthe most valuable and useful characteristic of thermostabilised GPCRs.SAH6 and SAH7 were also more tolerant to short chain detergents thanwild type SERT (FIGS. 5A-5B).

The affinity of [¹²⁵I]-RTI55 for both SAH6 and SAH7 is virtuallyidentical to the wild-type transporter (23), which strongly supports thecontention that the mutants are folded in a biologically relevantconformation. This is further supported by the cell surface expressionof both mutants in stable cell lines expressing either SAH6 or SAH7, asmisfolded SERT is retained in the endoplasmic reticulum (ER).Competition assays using both inhibitors and the substrate 5-HT providefurther evidence on the likely conformation that has been stabilised.Both SAH6 and SAH7 bind cocaine with higher affinity than wild-typeSERT. None of the mutations are in the region proposed to be theinhibitor binding site (30, 31) so in analogy to what has beenpreviously observed in GPCRs, these data suggest that SAH6 and SAE-17have been stabilised in a ‘cocaine-bound’ conformation. As cocaine hasbeen proposed to bind preferentially to the outward-open conformation ofSERT (32), it is likely that both SAH6 and SAE-17 are thermostabilisedin an outward-open state. The decrease in binding affinity of bothimipramine and paroxetine for SAH6 is consistent with thisinterpretation, as there are likely to be subtle differences between thebinding of these inhibitors compared to RTI55, even though they are allproposed to bind to the outward-open state (33, 34).

Example 5 Functional Activity of SAH6 and SAH7 Mutants

A characteristic of transporters is obviously their ability tofacilitate the vectorial movement of substrates across biologicalmembranes. 5HT transport catalysed by SAH6 and SAH7 was thereforecompared with the wild-type transporter in stable cell lines thatrobustly express the transporters on the cell surface of atetracycline-inducible HEK293 cell line (35, 36).

Although both SAH6 and SAH7 were capable of binding inhibitors and thesubstrate serotonin, no significant transport of [³H]-5HT into the cellwas observed in cell lines expressing cell surface-expressed SAH6 orSAH7, despite the presence of the mutants in the plasma membrane asdefined by confocal microscopy (FIGS. 8A-8B) suggesting that thereceptors are preferentially in one particular conformation. This is notdue to alterations in the binding site for 5HT, because 5HT prevented[¹²⁵I]-RTI55 binding in competition assays although the affinity for 5HTwas decreased by 1.5-2.9 fold. These data are consistent with the theorythat both SAH6 and SAH7 are thermostabilised in a specific outward-openconformation.

Example 6 Mapping of Thermostabilising Mutations to LeuT

There is a growing body of data which suggests that the structure ofSERT is very similar to that of the bacterial transporter LeuT.Structures of LeuT bound to antidepressant drugs have been determined,where the antidepressant drugs are also known to bind SERT (30, 31) anddespite the large difference in binding affinities (nM compared to nM),has led to plausible models for how antidepressant drugs inhibit SERT.We have therefore mapped the thermostabilising mutations identifiedherein to the structure of LeuT bound to sertraline (FIGS. 9-11C, FIG.12A-12D). Surprisingly it was found that all the mutations in SAH6 andSAH7 are found at the interfaces between transmembrane a-helices and.More specifically, in either a kinked region or in an a-helix adjacentto a kink. This suggests that mutations in specific regions of a proteincan be applied across a range of membrane proteins having similarthree-dimensional structures, thereby improving the probability ofobtaining conformationally stable mutants for use in crystallisation.

Example 7 Expression and Crystallisation of Thermostabilised SERT

A stable HEK293-GnTI⁻ cell line was constructed that expressed thethermostabilised serotonin transporter (SERT) to high levels. Oninduction with tetracycline, thermostabilised SERT was expressedsufficiently to allow the purification of 2 mg of transporter from 10 Lof cells using the detergent dodecylmaltoside. Crystals were produced byvapour diffusion methodology in 23% PEG400, 100 mM MES pH 5.9 and theydiffracted isotropically to about 8 Å resolution.

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1. A mutant membrane protein which has increased conformationalstability compared to its parent membrane protein, wherein the one ormore mutations are located at the interfaces between transmembranealpha-helices, or in a kinked region or in an alpha-helix adjacent to akink.
 2. A mutant membrane protein according to claim 1 which is aT-cell receptor complex, a growth factor receptor, a ligand-gatedtransmembrane ion channel, a voltage-gated transmembrane ion channel, atransmembrane transporter, an enzyme, a carrier protein, or an ion pump.3. A mutant membrane protein according to claim 2 which is atransmembrane transporter.
 4. A mutant membrane protein according toclaim 3 which is a transmembrane transporter and a member of theneurotransmitter sodium symporter family (NSS).
 5. A mutant membraneprotein according to claim 4 which is the cocaine-sensitive ratserotonin transporter (SERT) protein.
 6. A mutant membrane proteinaccording to claim 3 which is norepinephrine (NET), dopamine (DAT) orthe glycine transporter (GlyT).
 7. A mutant membrane protein accordingto claim 5 which comprises one or more amino acid mutations selectedfrom P499A, A505L, G113A, L99A, G278A, A169L, F311A, G115A, L405A andL406A.
 8. A mutant membrane protein according to claim 7 comprisingthree mutations selected from G278A, A505L, L99A and P499A.
 9. A mutantmembrane protein according to claim 7 comprising mutations L99A, G278Aand A505L.
 10. A mutant membrane protein according to claim 7 comprisingmutations L405A, P499A and A505L.
 11. A mutant membrane proteinaccording to claim 1 comprising at least one or more mutations which areat the corresponding amino acid positions of P499A, A505L, G113A, L99A,G278A, A169L, F311A, G115A, L405A and L406A as defined in the amino acidsequence of SERT shown in FIG.
 9. 12. A mutant membrane proteinaccording to claim 1 which has increased conformational stability whencompared to its parent to one or more of heat, a detergent, a chaotropeor an extreme of pH.
 13. (canceled)
 14. (canceled)
 15. A mutant membraneprotein according to claim 1 which is bound to a ligand. 16.-24.(canceled)